Neutropenia is a blood disorder characterised by an abnormally low number of neutrophil granulocytes. Neutrophils are active phagocytes (engulfers). Being highly motile, neutrophils quickly congregate at a focus of infection or inflammation. Neutrophils usually make up 50-70% of circulating white blood cells and serve as the primary defense against infections by destroying pathogens. Hence, patients with neutropenia are more susceptible to infections and without prompt medical attention, the condition may become life-threatening. Neutropenia can be acute or chronic depending on the duration of the illness.
The spectrum of neutropenia related infections has shifted in the past 20 years with fungal infections, particularly invasive moulds such as Aspergillus, Fusarium, and Zygomyces emerging as the principal infectious cause of mortality and morbidity. The incidence of invasive Aspergillus infection in patients undergoing allogeneic bone marrow transplantation (BMT) is approximately 15 percent, with mortality rates of 30 to 80 percent. Fusarium infection in these patients is fatal in 70 percent of cases.
In order to better understand the problems presented by neutropenia, it is helpful to understand some basic principles about blood cells, including their source and their development.
Blood cells develop from multipotent stem cells. These stem cells have the capacity to proliferate and differentiate. Proliferation maintains the stem cell population, whereas differentiation results in the formation of various types of mature blood cells that are grouped into one of the three major blood cell lineages, the lymphoid, erythroid and myeloid. It is the myeloid lineage, which is comprised of monocytes (macrophages), granulocytes (including neutrophils), and megakaryocytes, monitors the bloodstream for antigens, scavenges antigens from the bloodstream, fights off infectious agents, and produces platelets, which are involved in blood clotting.
Neutrophils differentiate from haematopoietic stem cells through a series of intermediate precursor cells, which can be distinguished by their microscopic morphological appearance, including such characteristics as the size of their nuclei, cell size, nuclear/cytoplasmic ratio, presence/absence of granules, and staining characteristics (See Atlas of Blood Cells: Function and Pathology, second edition, Zucker-Franklin et al.) Initially, the multipotent stem cell, gives rise to myeloid “progenitor cells” that generate precursors for all myeloid cell lines. The first myeloid progenitor is designated CFU-GEMM for “colony forming unit—granulocyte, erythroid, macrophage and megakaryocyte”. The CFU-GEMM progenitor, in turn, will give rise to a CFU-GM progenitor cell, which is otherwise known as a “colony forming unit—granulocyte macrophage”. In all of these descriptive terms, “colony” generally refers to a cell that is capable of giving rise to more than 50 cells as measured in 14 day in vitro assays for clonal growth. These cells will divide at least six times.
The CFU-GM is a committed progenitor: —it is committed to differentiation into granulocytes and macrophages only. It is neither capable of differentiating into other types of cells nor is it capable of dedifferentiating into earlier stage progenitor cells. The CFU-GM progenitor cell may then differentiate into a myeloblast. The time required for differentiation from a CFU-GEMM to a myeloblast is believed to be about 1-4 days. A myeloblast is the first of the series of cells that may be referred to as “precursors” to the neutrophils, as such cells, once allowed to fully develop (differentiate), can only form neutrophils, which it is believed, are only capable of undergoing fewer than six cell divisions and, therefore, do not form colonies in in vitro assays as described previously.
Once differentiation has progressed to the myeloblast stage, the myeloblasts undergo terminal differentiation. Myeloblasts differentiate into promyelocytes, which, in turn, differentiate into myelocytes over a course of about 4-6 days. Within another 5 days or so, myelocytes differentiate into metamyelocytes, which, in turn, differentiate into banded neutrophils. These banded neutrophils finally differentiate into mature, segmented neutrophils, which have a half-life of about 0.3 to 2 days.
During this progressive, morphological differentiation, changes in the surface antigens of these cells can be observed. Further, as neutrophil precursor cells differentiate, they lose their capacity to proliferate. In general, the less mature neutrophil precursor cells, namely the myeloblasts, promyelocytes, and myelocytes, retain their ability to proliferate. However, the more mature neutrophils, namely the metamyelocytes and the banded neutrophils, lose their capacity to proliferate, although they continue to differentiate into mature, segmented neutrophils.
The current treatment for chemotherapy induced neutropenia varies, but it typically involves dosage modulation or the cessation of the cytotoxic therapy along with the administration of granulocyte-colony stimulating factor (G-CSF) or other stimulating factors to increase the circulating neutrophil count. Peripheral blood contains approximately 10% of the body's neutrophil pool. Agents such as G-CSF act by causing the near immediate release of stored mature neutrophils and an increase in renewal and differentiation of stored progenitor and precursor neutrophils. It then takes the bone marrow around 10-15 days to replenish the neutrophil stores and thus the levels of circulating neutrophils, an effort made more difficult with myeloablative therapy which tends to destroy the progenitor and precursor cells. Therefore, even with the administration of G-CSF, patients are likely to benefit from supplemental neutrophil transfusion.
U.S. Pat. No. 6,146,623 describes a technique that several companies have tried or are trying in order to develop stem cell progenitor or precursor based therapies for the treatment of neutropenia. This involves isolating haematopoietic stem cells, expanding these cells ex vivo to a point where they are of a committed lineage, but are not fully differentiated, and then transfusing the expanded cells into the patient. An emphasis has been put on an expansion that produces cells that are predominantly late progenitors and precursors of mitotic neutrophil precursors and include CFU-GEMM, CFU-GM, myeloblasts, promyelocytes and myelocytes. One reason for this is that these cells are understood to retain their capacity to proliferate, whereas more mature neutrophils, namely the metamyelocytes, band neutrophils and segmented neutrophils, are post-mitotic cells and have lost their proliferative capacity. The guiding principle has been that these progenitors and precursors would differentiate while in circulation as well as engraft into the neutrophil compartment and begin to produce neutrophils, and hence the time period of neutropenia would be reduced. There are several disadvantages to this strategy; one is that the transfusion of mitotic progenitor and precursor cells may require tissue matching, to minimise the risk of graft-versus-host disease (GVHD). Another is that the most efficient stem cell expansion techniques reported only produce a 150-250 fold increase of the initial starting material, which makes these processes expensive, and lastly, that progenitors do not offer the needed immediate protection of mature, segmented neutrophils. Because of the disadvantages, these therapies tend to be expensive and still leave a patient at risk of infection for a significant period of time.
Another therapy, neutrophil transfusion, is currently a treatment option that is reserved only as a last option for the critically ill. This is due to a number of factors including the number of doses, the neutrophils needed per dose, the difficulty involved in recruiting matched donors, and the need to submit the donors to a mobilisation and aphaeresis procedure.
Currently neutrophils are collected from donors through an aphaeresis procedure with or without mobilisation. Mobilisation involves pre-treating the donor at least 12 hours in advance with steroids such as dexamethesone as well as granulocyte colony stimulating factor (G-CSF), a growth factor specific for neutrophils. Without mobilisation a donor is subjected to a 2-4 hour aphaeresis procedure that yields an average of 20×109 neutrophils. With mobilisation the same aphaeresis procedure yield is typically increased to 60-80×109 neutrophils and each collection constitutes a single dose.
It is not known how many of the collected neutrophils are viable and un-activated, but based on clinical reports it is suspected that greater than 70% provide no therapeutic value, most likely due to activation. Additionally, because the aphaeresis procedure does not completely filter out other blood and immune cells, the donation must be a blood type match to the recipient and there is an increased risk of alloimmunization as a result.
A possible alternative to collecting neutrophils from donors could be to produce neutrophils ex vivo. However, as mentioned above, the focus to date has been on producing mitotic precursors or progenitors for expansion in vivo. Further, existing techniques for producing mature neutrophils ex vivo by expansion of stem cells such as described in PCT application WO 03/080806 not only include contact with stromal cells, which can lead to contamination and problems for regulatory approval, but they are inadequate for providing the numbers of non-activated cells needed for clinical purposes. Cultures in vessels such as T flasks cannot be readily scaled up for production of clinical quantities of cells needed to treat neutropenia, whereas the use of larger scale methods such as bioreactors has not resulted in adequate levels of expansion of functional cells.
Accordingly there is a need for an improved ex vivo method for producing mature neutrophils in sufficient quantities to make possible the use of ex vivo expanded mature neutrophils on a clinical scale in therapies based on the transfusion of mature neutrophils into neutropenic individuals or those deemed at risk of developing neutropenia.